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Home , Macrophage, Macrophage elastase

Basic Research—Biology

The Expression of Macrophage and Elastases in Rat Periradicular Lesions Taisuke Morimoto, DDS, PhD,* Masahiro Yamasaki, DDS, PhD,* Kazuhiko Nakata, DDS, PhD,*,† Masahito Tsuji, DDS, PhD,* and Hiroshi Nakamura, DDS, PhD*,†

Abstract Macrophage elastase and neutrophil elastase are periradicularin- lesion evoked by bacterial infection consists of periapical inflamma- volved in tissue destruction in periradicular lesions.A tionThe with destruction of the periodontal and resorption of the alveolar purpose of this study was to examine these boneelastases and root1). (The periodontal ligament is a dense localized immunohistochemically during development of perira-between the cementum and the alveolar and supports the tooth. The destruction dicular lesions induced in rat mandibular first ofmolar the byapical periodontal ligament is initiated by degradation of the pulpal exposure for 7, 14, 21, 28, and 42 (ECM).days. Histo-E C composedM i s mainly of fibers, and elastic-system fibers in- logically, periapical developed from cluding7 to oxytalan fibers and -containing fibers (ie, elastic and fibers) 21 days and then subsided after 28 days. Theare areaubiquitous of components of this 2ligament ). Elastic-system ( fibers have been these lesions gradually increased from 7 to 28 investigateddays and in the rat molar periodontal ligament including its3 ). apical portion ( subsequently decreased at 42 days. Macrophage Theelas- of ECM degradation are both serine proteinases and matrix metallo- tase was first detected at 7 days and predominantlyproteinases (MMPs). shown from 14 to 28 days, whereas neutrophil elastaseMMPs are a family of zinc-dependent endopeptidases that possess the ability to gradually increased from 14 to 28 days. Macrophage degrade all components of the ECMs and play important roles in many physiologic and elastase was significantly greater than neutrophil elas- pathological processes4, ().5 Especially, MMP-2 (geratinase A), -7 (matrilysin), -9 tase from 7 to 21 days. These results suggest that (geratinase B), and -12 (macrophage metalloelastase) have elastinolytic properties macrophage elastase was enhanced from an early stage during the development of these lesions (and6). thatMMP-12 was discovered in thioglycolate-stimulated mouse peritoneal macro- neutrophil elastase was related to the expansionphages of7(). Murine MMP-12 complementary DNA was8), clonedand, subsequently,( periapical tissue destruction including bone resorption.human and rat homologues of MMP-12 complementary DNA 9 were–11). reported ( (J Endod 2008;34:1072–1076) MMP-12 has the ability to degrade ECM components by12 )attacking and otherelastin ( basement membrane components such as or collagen type V but not gelatins Key Words (13, 14). Collagen type IV or laminin is also degraded by murine or human MMP-12 Elastase, macrophage, neutrophil, periradicular lesion,(13, 14) but not by rat 11MMP-12). Macrophages ( with those proteolytic activities are rat able to penetrate basement membranes and invade 14several). Varioustissues roles( of MMP-12 in disease models have been revealed to date, such as the development of emphysema15 ),( prevention of tumor growth through angiostatin generation (16, 17), and degeneration of the aortic medium in abdominal18 ).aortic aneurysm ( From the *Department of Endodontics and †Research In- By using the complementary DNA microarray analysis, MMP-12 in rat periradicular stitute of Advanced Oral Science, Aichi-Gakuin University lesion was upregulated compared with healthy19 ). controlBut, to( date, few reports School of Dentistry, Nagoya, Japan. Supported by “AGU High-Tech Research Center” Project have been published with respect to the role of MMP-12 in periodontal and periradicu- for Private University: matching fund subsidy from MEXT (Min- lar tissues. istry of Education, Culture, Sports, Science and Technology), Neutrophil elastase, which is involved in the degradation of elastin, is one of the 2003–2007. Address requests for reprints to Dr. Hiroshi Nakamura, serine proteinases and it has strongly degradative action toward several proteins. Se- Department of Endodontics, School of Dentistry, Aichi-Gakuin creted cathepsin G and proteinase 3 also have an elastinolytic property in serine pro- University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651 teinase 20(). Polymorphonuclear leukocytes (or ) contain an abundance of Japan. E-mail address: [email protected]. lysosomal granules filled with proteolytic enzymes and other components. Neutrophil 0099-2399/$0 - see front matter Copyright © 2008 American Association of Endodontists. granules can be differentiated into specific and azurophilic (or primary) granules, the doi:10.1016/j.joen.2008.06.012 latter containing the serine proteinases such as elastase and cathepsin G as well as other enzymes 21(). Neutrophil elastase secreted by activated neutrophils can bind to and degrade ECM. This has wide substrate specificity and has strong competency in degrading a variety of the proteins including22). Thereelastin is( a possibility that it evokes the severe tissue destruction seen 23in) pulpitisand periradicular ( lesions (24). In human periapical disease, neutrophil elastase levels of periapical associated with clinical symptoms25) and( there may be a possibility that it serves as a diagnostic marker (26). The purpose of the present study was to examine the involvement of macrophage elastase and neutrophil elastase in the development of periradicular lesions in rats.

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Material and Methods primary polyclonal reactive with macrophage elastase The Induction of Periradicular Lesions (sc-8839) and neutrophil elastase (sc-9521) were obtained from Santa Twenty-four male Wistar rats, weighing about 250 g, were used in Cruz Biotechnologies (Santa Cruz, CA). Normal goat serum was used as the present study. Periradicular lesions were induced as described pre- the negative control. The sections were stained by use of ABC Staining viously (27, 28). In brief, all animals were anesthetized, and the pulpal System (sc-2018, Santa Cruz Biotechnologies). The slides were exposed tissues in the left and right mandibular first molars were exposed so that to hydrogen peroxide in deionized phosphate-buffered saline (PBS) for perforation of the furcation would be avoided. The exposed areas were 10 minutes to quench the endogenous peroxidase activity and subse- left open to the oral environment until the animals were killed. quently reacted with 1.5% goat serum diluted in PBS for an hour to block nonspecific binding. These slides were incubated with Tissue Preparation and each primary antibody for 12 hours, followed by biotinylated antibody Animals were sacrificed at 7, 14, 21, 28, and 42 days (each period, for 30 minutes. Then, they were incubated with avidin-biotinylated per- n ϭ 4) after the pulpal exposure. Animals without the exposure were oxidase complex for 30 minutes. For the final chromogenic reaction, used as the control (day 0, n ϭ 4). At the time of sacrifice, the mandi- the slides were exposed to the substrate solution consisting of diamino- bles of all animals were removed and fixed in periodate-lysine-parafor- benzidine-tetrahydrochloride and hydrogen peroxide and were coun- maldehyde solution for 24 hours at 4°C. The mandibles were decalci- terstained with hematoxylin. The periapical tissue of the mesial root of fied by being soaked in 5% EDTA solution until complete decalcification the mandibular first molar was examined for both -positive cells. (approximately 60 days) at 4°C. Then, these were embedded in OCT compound (Miles Scientific, Naperville, IL) and sectioned serially at 5 Histometry ␮m in the mesiodistal plane. Quantitative analysis was performed on 4 serial sections from each The presence of macrophage elastase and neutrophil elastase molar. The area of the periradicular lesion was measured histometri- in the periapical tissue were examined immunohistochemically. The cally as described previously (26, 27). The area of the lesion was the

Figure 1. Immunohistochemical staining for macrophage elastase. (A) In the mandibular first molar at 7 days after the exposure, some elastase-expressing cells detect in the residual pulpal tissue and around the root apex. B is a high-magnification view of cropped area in A. These cells gradually increase at (C) 14 and (D) 21 days. (E) By 28 days, these cells increase in number and are observed in and around the abscess. F is a high-magnification view of cropped area in E. G is a cropped area in F. The arrows indicate macrophage elastase-positive cells. These cells are round-shaped in high magnification. By 42 days, these cells decrease in number, but some still remain in the abscess (H). A, C, D, E, and H scale bars ϭ 1.0 mm. B and F scale bars ϭ 0.25 mm. G scale bar ϭ 0.025 mm.

JOE — Volume 34, Number 9, September 2008 Expression of Elastases in Rat Periradicular Lesions 1073 Basic Research—Biology periodontal ligament between the root apex and alveolar bone in the flammation and alveolar bone resorption were observed. By 21 days, a mesial root. The cells reactive with antimacrophage and antineutrophil small abscess was found around the root apex. Severe inflammation and elastases were counted in an area 0.6 mm square of the periapical bone resorption were investigated. By 28 days, the abscess had ex- portion surrounding the root apex of the mesial root, and the number of tended. Resorption of the apical root and alveolar bone was evident at antigen-positive cells per unit area (cells/mm2) was calculated (28). this time. The abscess and inflammation in periapical tissue had de- creased by day 42. Statistical Analysis and Animal Protocol The results of all measurements were presented as mean values Ϯ Macrophage Elastase standard deviations. The average values were determined for each ani- No macrophage elastase-expressing cells were present in the peri- mal, and the average values were calculated for each group. The statis- apical tissue at day 0. At 7 days after the exposure, however, a few tical significance of differences was determined by the Mann-Whitney U elastase-expressing cells were detected around the root apex (Fig. 1A test. The principles of laboratory animal care according to the animal and B). By 14 days, these cells had increased in number (Fig. 1C). At 21 protocol institutionally approved by the ethics committee of School of days, such cells were observed in and around the apical abscess (Fig. the Dentistry of Aichi-Gakuin University were followed. 1D). By 28 days, many cells were seen there (Fig. 1E, F, and G). By 42 days, these cells decreased in number, but some still remained (Fig. 1H). Results Histologic Findings Neutrophil Elastase In the mandibular first molar with no exposure, the periapical No neutrophil elastase-expressing cells were observed in the peri- tissue did not change. At 7 days after the exposure, a few inflam- apical tissue at day 0 or 7 days (Fig. 2A). At 14 days, a few elastase- matory cells were noted in the periapical tissue. At 14 days, slight in- expressing cells were detected around the root apex (Fig. 2B and C). By

Figure 2. Immunohistochemical staining for neutrophil elastase. (A) At 7 days, a few neutrophil elastase-expressing cells are observed in the residual pulpal tissue, whereas no cells are present in periapical tissue. (B) By 14 days, antigen-positive cells detect in apical periodontal ligament. C is a high-magnification view of cropped area in A. These cells increase in number from (D)21to(E) 28 days. At 28 days, these scatter in apical periodontal ligament. F is a high-magnification view of cropped area in E. G is the cropped area in F. Arrows indicate the macrophage elastase-positive cells. Arrows point to the neutrophil elastase-positive cells. (G) These cells are polymonophic shaped and polynuclear in high magnification. (H) By 42 days, their number decrease, but some remain in the apical abscess. A, B, D, E, and H scale bars ϭ 1.0 mm. C and F scale bars ϭ 0.25 mm. G scale bar ϭ 0.025 mm.

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TABLE 1. Changes in Mean and SD of the Area of the Periradicular Lesion and present study, macrophage elastase first appeared in the periradicular the Number of the Macrophage and Neutrophil Elastase-expressing Cells per lesion at 7 days and predominated at 14, 21, and 28 days. The most Unit Area evident increase in OX6-positive cells (macrophages) in this lesion was Area of Macrophage Neutrophil observed at an early stage postoperatively (29). When macrophages are Experimental Periradicular Elastase Elastase activated, they may participate in triggering the development in this Period Lesion (mm2) (cells/mm2) (cells/mm2) lesion (30). Dendritic/Langerhans cells, which are positive for ED1 (a Day 0 0.229 Ϯ 0.110 0.88 Ϯ 0.85 1.20 Ϯ 0.62 general marker of mononuclear ), can release macrophage Day 7 0.264 Ϯ 0.089 13,70 Ϯ 2.91 2.21 Ϯ 13.70 elastase (31). Because macrophages are activated in the initial stage of Day 14 0.437 Ϯ 0.087 24.60 Ϯ 3.78 4.30 Ϯ 3.60 periradicular lesion, macrophage elastase may contribute to the early Ϯ Ϯ Ϯ Day 21 0.776 0.141 62.00 10.20 42.90 6.62 destruction of ECM in periapical tissue. Day 28 1.038 Ϯ 0.116 84.20 Ϯ 16.40 73.20 Ϯ 19.60 Day 42 0.832 Ϯ 0.096 49.30 Ϯ 13.90 53.10 Ϯ 17.40 Neutrophil migration and degranulation in the affected regions of inflamed pulp require the actual presence of . After the bacterial infection has spread beyond the apical foramen, activated neutrophils assemble in the periapical tissue and phagocytize the bacteria. Neutro- 21 days, these cells had increased in number and were seen in the apical phil has the defense mechanism involved in the killing of invading bac- abscess (Fig. 1D). By 28 days, these cells were still observed there (Fig. teria by the liberation of reactive species (32) and lysosomal 2E, F, and G). At 42 days, these cells were fewer in number, but some enzymes (20). In these serine proteinases, elastase, cathepsin G, and still remained (Fig. 2H). proteinase 3 have elastinolytic activity. Especially, this elastase that is a strong can kill bacteria (25). It may secrete neutrophil elastase Histometrical Result in the periapical lesion as result of their degranulation with other serine The area of periradicular lesion and the numbers of macrophage and proteinases. A positive relationship exists between and the in- neutrophil elastase-expressing cells are presented in Table 1. The area crease of neutrophil elastase activity in humans (23). Polymorphonu- increased gradually from 0 to 28 days after pulpal exposure, whereas it clear leukocyte elastase was observed in the periapical tissue of patients decreased by 42 days. There was no significant difference in area be- clinically diagnosed as having irreversible pulpitis (24). Neutrophil tween the control rats without exposure and those rats with pulpal elastase is detected in the periodontal ligament of patients with peri- exposure for 7 days. This area from 14 to 42 days was significantly larger odontitis (33). Although the neutrophil elastase was already known to than the control one (p Ͻ 0.05). Especially, this area showed the be increased in the inflammation of other tissues, the increase was greatest enlargement from 14 to 21 days. investigated in the periapical tissue with inflammation. The macrophage The number of macrophage elastase-expressing cells in the peri- elastase was first detected at 7 days, whereas the neutrophil elastase was apical tissue increased gradually from 7 to 28 days after pulpal expo- observed in and around the apical abscess after 14 days. Neutrophil sure, whereas it decreased at 42 days. Their numbers from 7 to 42 days appeared later compared with macrophage elastase. These results sug- were significantly larger than the control one (p Ͻ 0.05). Few neutro- gest that the expansion of this lesion with alveolar bone destruction may phil elastase-expressing cells were detected at 7 and 14 days after the follow the increase of neutrophil elastase. exposure. The number of these cells increased gradually from 21 to 28 In conclusion, these investigations have indicated that macro- days, whereas it decreased at 42 days. There was not a significant dif- phage elastase activity enhanced from an early stage during the devel- ference in the number between the rats without exposure (control) and opment of periradicular lesions and that neutrophil elastase activity was those exposed for 7 or 14 days, but the numbers for the animals with related to the expansion of periapical tissue destruction including bone pulpal exposure for 21 to 42 days were significantly larger (p Ͻ 0.05). resorption. 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